Trypanosoma congolense causes the most economically important animal trypanosomosis in Africa. In South Africa, a rinderpest pandemic of the 1890s removed many host animals, resulting in the near-eradication of most tsetse species. Further suppression was achieved through spraying with dichlorodiphenyltrichloroethane (DDT); however, residual populations of Glossina austeni and G. brevipalpis remained in isolated pockets. A total of 506 of these tsetse flies were captured in the Hluhluwe-iMfolozi Park, the St Lucia Wetland Park and Boomerang commercial farm. The polymerase chain reaction (PCR) was used to determine the infection rate and frequency of mixed infections of these flies. Additionally, 473 blood samples were collected from cattle at communal diptanks and a commercial farm in the area and each one examined by the haematocrit centrifugation technique (HCT). Furthermore, buffy coats from these blood samples were spotted onto FTA Elute cards and the DNA extracted from each one tested using 3 separate PCRs. The HCT revealed the presence of trypanosomes in only 6.6 % of the blood samples; by contrast, species-specific PCR detected trypanosome DNA in 50 % of the samples. The species-specific PCR detected trypanosome DNA in 17 % of the tsetse flies, compared with the nested PCR targeting rDNA which detected trypanosome DNA in only 14 % of the samples. Over time, the transmission of Savannah-type T. congolense and Kilifi-type T. congolense as mixed infections could have an impact on disease manifestation in different hosts in the area.